Over 20 years of research on the function of the HIV rev gene, has demonstrated that a major function of Rev, in conjunction with the RRE, is to mediate the nucleo-cytoplasmic export of the unspliced or incompletely spliced viral RNA. Export of unspliced RNA is a task that all retroviruses have to perform. However, many less complex retroviruses accomplish this efficiently, simply by having an RNA element in their genome that interacts exclusively with cellular proteins. There is very little data to effectively the answer the question of why HIV has evolved Rev. This exploratory proposal will address the question of whether Rev and the RRE co-evolve in an HIV infected individual, and whether this could allow Rev to play a key role in HIV pathogenesis that we have not yet fully uncovered. This proposal explores the idea that many different Rev and RRE alleles, each having different levels of function, may be expressed in infected individuals over time and/or at the same time in different compartments. If this is true, then Rev could serve as an important modulator of infection, acting to fine tune replication levels at different stages of disease, and also help the virus evade the immune system. There are two specific aims: Specific Aim #1 To analyze Rev and RRE Variation and Function in Infected Individuals. Single genome sequencing will be used to determine cognate Rev and RRE sequences both in newly infected and chronically infected individuals. We will determine how Rev and RRE sequence and function evolves over time and if there is covariation. The functional activity of these Rev and RRE sequences will also be analyzed. We anticipate that these studies will pinpoint regions of variability and of invariance in both rev and RRE. From this data, we will be able to determine the full nature and extent of Rev and RRE variation in infected individuals, and its effect on function, and also if any trend emerges that could provide clues into a role for Rev as a mediator of pathogenesis. Specific Aim #2: Selection of Functional RRE Sequences with or without Envelope Protein Coding Constraints As a complement to the studies in aim #1, in this aim, we will randomize portions of the RRE to identify sequences and structures that the RRE must maintain to be functional. We will do this both under conditions where the RRE sequence is constrained by the need to allow coding for a functional envelope protein and when it is not. Our efforts will initially focus on the Stem Loop III region, which are outside of the primary Rev binding site, but overlapping with HR2 of gp41. Predictions of functional RRE sequence and structure made from the DNA sequence analysis will be confirmed in functional and structural studies. PUBLIC HEALTH RELEVANCE: This proposal will define the limits of variation of the HIV rev gene, and its associated RNA element, the RRE, in infected individuals. This is being done as a first step towards identifying clues into a role for Rev as a mediator of pathogenesis. The experiments in this proposal will give further insight into the possibility of using Rev as a novel target for HIV therapy.